The invention pertains to the field of liquid chromatography, and, more particularly, to the field of pumping systems to control the flow of solvent and sample through liquid chromatography columns.
Liquid chromatography is a process whereby the known components of a sample may be analyzed to determine the quantity of each component in a sample of unknown composition. To make such an analysis, the sample is dissolved in a solvent stream which is then passed through the liquid chromatography column. Liquid chromatography columns contain particles often modified with chemical reagents which act upon the solvent and sample system to retain the various sample components for different amounts of time. In other words, as the sample is pumped through the column, the output stream is comprised of the basic solvent which carries the sample plus the individual components of the sample which emerge from the output of the column at different times.
Liquid chromatography analysis systems detect the presence of the various components of the sample in the output stream using various types of detectors. The purpose of these detectors is to record characteristics of the output streams and to generate signals in the form of output waves (spikes) which represent the presence and quantity of each particular sample component. Each of these spikes appears in the output stream at a different time. The time of occurrence of each spike indicates which component of the sample is then emerging. These "retention" times are based upon the known characteristics of each sample component and the known characteristics of the liquid chromatography column in acting upon that component. However, these retention times are also based upon assumptions as to the magnitude of the flow rate of solvent through the column and the relative stability of this flow rate.
Normally, light absorbance detectors are used at the output of the liquid chromatography column to detect changes in light absorbance as sample components emerge. These absorbance detectors depend upon the light absorbance of the sample components in the output stream and generate signals indicative of this absorbance which can be used to quantify the size of the peak.
However, in certain applications, the sample components do not absorb light. In these applications, detectors such as refractive index, fluorescence, conductivity, electrochemical and other types of detectors are used to quantify the peaks. Refractive index detectors measure the varying amounts of refraction of a light beam as it passes through the output stream. The amount of refraction or bending of the light beam at any particular time as it passes through the output stream depends upon the refractive index and the magnitude of a particular sample component in the output stream at that time. These detectors output signals which quantify the amount of refraction.
An unfortunate side effect of the use of refractive index detectors is that they are very sensitive to changes in the composition of the solvent carrier. In many applications, it is useful to have solvent compositions with multiple components, and frequently "gradient" solvents are desirable. A gradient solvent is a solvent comprised of two or more solvents where the solvent composition is varied over time. A very popular way of making up gradient solvents is to have each solvent delivered by a separate pump and to run each pump at an appropriate speed to make up the currently desired solvent composition.
Gradient solvent compositions are not used in the prior art where refractive index detectors must be used. Additionally, the use of pump blended solvent mixtures cannot occur with refractive index detectors. The only current method of forming the solvent composition for use with refractive index detectors is to form the solvent composition in a separate container and mixing it thoroughly. This separate process of forming the solvent composition and mixing it thoroughly causes the relative makeup of the solvent composition to be known and non-varying.
However, this is an inconvenient process for use in commercial liquid chromatography systems and is incompatible with creation of a continuously changing gradient. In commercial liquid chromatography systems, solvent composition makeup is normally done in either of two ways. One way involves using a plurality of valves which gate the various solvent components into the pump which drives the solvent composition through the liquid chromatography column. Another way is through the use of multiple pumps as described above.
However, the reproducibility of the solvent composition which can be formed using either the multiple pump or valve method is not sufficient for use with refractive index detectors. This is because the pumps or valves cannot control the exact composition of the solvent mixture as well as by hand mixing. Most pumps have a repeatability factor of 0.1% which is not high enough for use with refractive index detectors.
Basically, refractive index detectors are so very sensitive to the solvent composition that even the slightest error in the relative magnitude of the quantities of solvent components in the solvent mixture will lead to a phenomenon called "baseline drift". Baseline drift refers to changes in the output signal of the detector which are caused by changes in the solvent composition and not changes in the sample component content of the output stream. Basically, baseline drift is noise which degrades the accuracy of the results which can be obtained by a liquid chromatography system. In an ideal system, the output of the detector at the output of the liquid chromatography column would be a steady state stable value when no sample was injected into the input stream entering the column. However, when solvent makeup valves or individual pump motors for each solvent component are used to make up the solvent composition, the errors in the composition which result cause the baseline to drift erratically because of the extreme sensitivity of the refractive index detectors to the slight changes in the refractive index of the solvent composition itself. The same problem can occur with use of other detectors, but is aggravated when using refractive index detectors.
Another problem with the use of pumps for solvent makeup is that the solvent composition is often not thoroughly mixed. This results in erratic baseline drift also.
Further, with absorbance types of detectors, the absorbance of the solvent itself changes at different wavelengths. This too can result in baseline drift error.
One solution to baseline drift that has been tried in the prior art is to use an insensitive range on the recorder used to record the results of a run. Unfortunately, this limits the sensitivity and resolution of the system. Thus, the use of gradient solvents with refractive index detectors has not been possible in the past and the use of pumps for solvent makeup has not been possible when using refractive index detectors even when gradients were not being used. Further, baseline drift is also a problem with other types of detectors such as absorbance detectors where the wavelength of the light is sensitive to the absorbance characteristics of the solvent components.
Others have attempted to solve the problem of baseline drift in the prior art but have failed. At least two groups of workers in the art have tried dual flow chromatography systems whereby the stream of solvent is split by a T connection in front of the column into two paths. One of these two paths was routed through the column while the other flow path was coupled directly to the common detector reference flow path input. This approach is described by Hunkapillar and Hood in Science magazine, Vol. 207, p. 24 (1980) and by Stevenson and Burtis in Clinical Chemistry, Vol. 17, page 774 (1971). The problem with this approach is that no positive displacement means is provided for each path. Thus, if differences in the resistance to flow exist between the two flow paths, unequal flow rates exist in each path, and the reference signal will become "out of synchronization" with the sample signal for which the reference signal is supposed to act as a reference. This cause errors.
Accordingly, there has arisen a need for a liquid chromatography system which is free of baseline drift in all applications. Such a system should be able to mix solvent gradients using pumps or solvent makeup valves to make either gradient or constant solvent compositions and be usable with any type of detector including refractive index detectors without errors caused by baseline drift.